CN106497888B - Salmonella bacteriophage and bacteriophage bactericidal composition and its application - Google Patents
Salmonella bacteriophage and bacteriophage bactericidal composition and its application Download PDFInfo
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- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
- C12N2795/10121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
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- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
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Abstract
The invention discloses salmonella bacteriophage and bacteriophage bactericidal composition and its applications, bacteriophage provided by the invention is Salmonella enteritidis bacteriophage LPSE1 and salmonella typhimurium bacteriophage LPST10, either bacteriophage LPSE1 or LPST10, can have good inhibiting effect to salmonella, its tolerance to pH and temperature is preferable simultaneously, application example in food system also confirms that have good effect when it is used as in bacteriostatic agent.After two kinds of bacteriophages are mixed, find there is synergistic effect between bacteriophage, there is more useful inhibiting effect compared to single bacteriophage.
Description
Technical field
The present invention relates to field of food safety, and in particular to salmonella bacteriophage and bacteriophage bactericidal composition and its should
With.
Background technology
It is shown according to the food origin disease detection architecture data in China, the food-safety problem as caused by food-borne microorganism
Shared composition ratio is up to 43%.And 70%-80% is wherein accounted for by salmonellal food origin disease outburst quantity.Last decade
(2004-2015) U.S. food origin disease monitoring net each department food origin disease breaks out report display, and global salmonella causes
Food origin disease breaks out rate and rises year by year, breaks out and ranks first.And salmonella is then included in and has by the World Health Organization (WHO)
It seriously endangers the food transmission venereal disease opportunistic pathogen with medium harm and has set up global salmonella monitoring net (WHO-GSS), strengthen
To salmonella-polluted monitoring.Salmonella is widely distributed, and serotype up to more than 2500 is planted, with salmonella typhimurium and
The case that this 2 kinds of Bacterium enteritidis has humans and animals pathogenic salmonella to cause salmonellosis is most, multiple
In summer and autumn.Blood can be entered by intestinal epithelial cell and cause septicemia, and generate endotoxin, cause acute gastroenteritis, food
The diseases such as object poisoning.Meat and meat products, egg products, dairy products, aquatic products and quick-frozen food are Foodborne salmonellas in food
The main source polluted in system.It in addition, may be in food transport, storage and process operation process by more than raw material or product
Cross contamination is brought to other products such as fruits and vegetables, increase cause of disease diffusion and causes the risk of infection.
At present for the control of food borne pathogens, can substantially be controlled two stages：For " before the harvest " stage,
Namely for producing poultry product, the animal of dairy products, mainly using chemicals (antibiotic and bacteriostatic compound), vaccine
The methods of cause of disease positive livestock and poultry are isolated to play the role of prevention and control cause of disease in immune or rejecting；For " after the harvest " stage
Product, that is, food material and product, mainly by chemical method (such as：Disinfectant, antibiotic and antibacterial substance) and
Physical method is (such as：Heating, ray and low temperature) eliminate or control cause of disease.
Addition compound carries out antibacterial chemical method and has received in worldwide at present greatly to query and very
To forbidding.Such as, 2006, European Union disabled antibiotic comprehensively and growth promoter uses in animal and fowl fodder.Lead in physical method
The safety for crossing the method that ray is sterilized always exists dispute, while need special installation, and application also suffers from querying.Most
Common thermal sterilization method can not but be applied in certain raw-food materials, semi-finished product or product, such as need to keep fresh
Raw material livestock meat, fresh fruits and vegetables and fresh egg and liquid eggs, they are improper before consumer and processing ground is sent to pass through
Heat treatment sterilization.Cause of disease can only be inhibited by the method for keeping low-temperature treatment more than raw-food material or finished product to a certain extent
Growing multiplication cannot but kill cause of disease, and under suitable conditions, they still have infection and lead to the risk of cross contamination.
It begins attempt to prevent using bacteriophage and control foodborne bacterial pathogens in the world at present.For example, 2006, the U.S.
Food and medicine Surveillance Authority (FDA) ratifies ListShieldTMBacteriophage product can be used as food additives, for ready-to-eat food
Listeria monocytogenes is controlled to pollute in birds meat products.Bacteriophage is a kind of energy specific infection bacterium and proliferation speed
Spend fast virus.Wherein virulent phage is by specific recognition host strain bacterium, the amount reproduction and final in host bacterial
Cracking discharges a large amount of progeny phages and plays antibacterial effect.
This research group has developed a kind of method using bacteriophage prevention and control to prevent and control salmonella.Bacteriophage has
It has the advantage that so that it can play a role in salmonella communication process is prevented：
(1) its high specificity does not influence body normal flora；
(2) small molecule environmentally protective, that bacteriophage is made of protein and nucleic acid, it is not heterologous for human or animal
Substance, the metabolic process entered in human or animal's body can be considered as natural process, environmentally safe；
(3) first use can realize multiple prevention, and bacteriophage can realize self-reproduction, in animal still in product
On can be achieved first use can realize multiple prevention；
(4) portability is strong.It is prepared into after preparation, needs not rely on special instruments and equipment and condition, do not need to be special
Conservation environment.
(5) have a wide range of application, do not limited by application and place.It can be applied to livestock and poultry cultivation process and place, livestock and poultry
Salughtering equipment and place, food material transporting equipment, food processing equipment, food material, semi-finished product and finished product.
Invention content
The object of the present invention is to provide a kind of salmonella bacteriophage LPSE1, and the salmonella bacteriophage is
It send to China typical culture collection center preservation, Classification And Nomenclature：Salmonella enteritidis bacteriophage (Salmonella
Enteritidis bacteriophage) LPSE1, deposit number is：CCTCC NO：M 2016472, preservation date 2016
On September 9, address：Wuhan, China Wuhan University.
The object of the present invention is to provide a kind of salmonella bacteriophage LPST10, and the salmonella bacteriophage is
It send to China typical culture collection center preservation, Classification And Nomenclature：Salmonella typhimurium bacteriophage (Salmonella
Typhimurium bacteriophage) LPST10, deposit number is：CCTCC NO：M 2016473, preservation date 2016
On September 9, address：Wuhan, China Wuhan University.
It is also an object of the present invention to provide a kind of salmonella phage mixture, which includes sramana
There is synergistic effect between two kinds of bacteriophages of the composition, have to salmonella well in Salmonella bacteriophage LPSE1 and LPST10
Inhibiting effect.
It is also an object of the present invention to provide a kind of application of salmonella bacteriophage LPSE1, including this is bitten
Thalline is used to inhibit pollution or the preparation of Salmonella in Food for inhibiting the drug of salmonella.
It is also an object of the present invention to provide a kind of applications of salmonella bacteriophage LPST10；Including this is bitten
Thalline is used to inhibit pollution or the preparation of Salmonella in Food for inhibiting the drug of salmonella.
The last one has been designed to provide a kind of application of salmonella phage mixture to the present invention, including this is bitten
Thalline is used to inhibit pollution or the preparation of Salmonella in Food for inhibiting the drug of salmonella.
In order to achieve the above object, the present invention takes following technical measures：
Inventor isolated two plants of salmonella bacteriophages from sewage, are respectively designated as salmonella bacteriophage
LPSE1 and salmonella bacteriophage LPST10.
The salmonella bacteriophage LPSE1 has been sent to China typical culture collection center preservation, Classification And Nomenclature：Intestines
Scorching salmonella bacteriophage (Salmonella enteritidis bacteriophage) LPSE1, deposit number are：CCTCC
NO：M 2016472, preservation date are September in 2016 9 days, address：Wuhan, China Wuhan University.
The salmonella bacteriophage LPST10 has been sent to China typical culture collection center preservation, Classification And Nomenclature：
Salmonella typhimurium bacteriophage (Salmonella typhimurium bacteriophage) LPST10 preserving numbers are：
CCTCC N O：M 2016473, preservation date are September in 2016 9 days, address：Wuhan, China Wuhan University.
Two kinds of bacteriophages can form larger bright plaque, edge clear rule on solid plate.Its pnagus medius
LPSE1 can be formed with 2mm plaques, and bacteriophage LPST10 on solid plate diameter in 0.5mm or so.
The bacteriophage LPSE1 and LPST10 is single use or is used in mixed way, and could be used for preparing food
Bacteriostatic agent or salmonella inhibitor.
Concrete scheme refers to specific embodiment.
Compared with prior art, the present invention has the following advantages：
(1) the bacteriophage stoste potency after enriched is high.In the present invention, the potency of bacteriophage LPSE1 and LPST10 >=
109pfu/mL。
(2) at different conditions, it is strong to split bacterium ability.In the present invention, bacteriophage LPSE1 and LPST10 has been respectively compared to exist
Under conditions of MOI=10, MOI=1, MOI=0.1, MOI=0.01, MOI=0.001 to Bacterium enteritidis ATCC13076 and
The bacteriostasis of salmonella typhimurium ATCC14028 is concurrently respectively provided with certain activity under the conditions of now different MOI.
(3) pH ranges are wider.In the present invention, the pH stability boundaries 3-13 of bacteriophage LPST10；Bacteriophage LPSE1's
PH stability 4-12, either LPST10 exclusive use or the two, which are combined, makes have the wider pH scope of applications.
(4) thermal stability is good.In the present invention, bacteriophage LPST10 and LPSE1 is basically unchanged in 30-60 DEG C of potency.
In conclusion this biological agent, which includes, can effectively inhibit, crack the bacteriophage of salmonella, said preparation can not only be
Using to prevent salmonella when animal is in condition of living organism, raw-food material can also be become in animal or product is answered
With, can also be applied to cultivation and food life processing site.
Description of the drawings
Fig. 1：Salmonella bacteriophage LPSE1 double-layer plate plaque figures.
Fig. 2：Salmonella bacteriophage LPST10 double-layer plate plaque figures.
Fig. 3：Bacteriophage LPSE1 cracks the host range positive findings figure of different serotypes salmonella；
Wherein：C represents SJTUF10984, figure in B expressions SJTUF10978, Fig. 3 in A expressions ATCC13076, Fig. 3 in Fig. 3
F represents CMCC50094 in E expressions ATCC13311, Fig. 3 in D expressions ATCC14028, Fig. 3 in 3.
Fig. 4：Bacteriophage LPST10 cracks the host range positive findings figure of different serotypes salmonella；
Wherein：C represents SJTUF10984, figure in B expressions SJTUF10978, Fig. 4 in A expressions ATCC13076, Fig. 4 in Fig. 4
F represents CMCC50094 in E expressions ATCC13311, Fig. 4 in D expressions ATCC14028, Fig. 4 in 4.
Fig. 5：The Electronic Speculum observation figure of salmonella bacteriophage LPSE1.
Fig. 6：The Electronic Speculum observation figure of salmonella bacteriophage LPST10.
Fig. 7：The growth curve of salmonella bacteriophage LPSE1.
Fig. 8：The growth curve of salmonella bacteriophage LPST10.
Fig. 9：Under the conditions of MOI values are 10, bacteriophage LPSE1 and LPST10 composition is to Bacterium enteritidis and mouse typhus
Salmonella splits bacterium capability result figure.
Figure 10：Different pH are to the exercising result figure of bacteriophage LPSE1.
Figure 11：Different pH are to the exercising result figure of bacteriophage LPST10.
Figure 12：Different temperatures is to the exercising result figure of bacteriophage LPSE1.
Figure 13：Different temperatures is to the exercising result figure of bacteriophage LPST10.
Specific embodiment
It is further illustrated the present invention with reference to embodiment, but the scope of protection of present invention is not limited to implement
The range of example statement.
The preparation method of 2 × YT culture mediums is in embodiment：Peptone 1.6g, yeast extract 1.0g, NaCl 0.5g, adds
Distilled water is to 100mL.
Embodiment 1:
Screening, purifying and the preservation of salmonella bacteriophage
1st, the screening of salmonella bacteriophage
Sewage sample 10mL is taken, is filtered with a diameter of 0.22 μm of millipore filter.It is added separately to equipped with 2 × YT of 20mL
It is another to add in culture to the sensitive indicator bacterium solution 5mL of logarithmic phase in the 50mL sterilizing triangular flasks of culture medium.37 DEG C of shaken cultivations
12-18h makes bacteriophage that may be present in sample achieve the effect that proliferation enrichment.
By above-mentioned culture solution in 50mL centrifuge tubes, at 4 DEG C, 8000 × g centrifugation 15min take supernatant with 0.22 μ L
Membrane filtration.The culture medium and exponential phase host strain bacterium solution for adding in sterilizing according to the method described above are enriched with three times repeatedly.Point
Not Cai Yong point sample method, rubbing method preliminary identification：There is apparent plaque sample further using double-layer agar technique, through gradient dilution, to see
Plaque morphology is examined, finally obtains two plants of bacteriophages, respectively bacteriophage LPSE1 and bacteriophage LPST10, as a result such as Fig. 1 and figure
Shown in 2.
2nd, the Sterility testing of bacteriophage stoste
Take bacteriophage separating obtained 8000 × g of stoste centrifugations 15min, 4 DEG C；Supernatant carefully is taken, with 0.22 μm of membrane filtration.
Filtrate is taken to be spread evenly across on solid agar tablet, cultivates whether there is bacterium colony on observation tablet after 12h in 37 DEG C.
3rd, the amplification cultivation of bacteriophage and purifying
Using solid multiplication method, picking is relatively independent in the double-layer plate of culture bacteriophage stoste, big and the smooth of the edge
Plaque, be inoculated in 1mL 2 × YT fluid nutrient mediums, 37 DEG C, 200r/min shaken cultivations 8h or so.At 4 DEG C, 8000 ×
G centrifuges 15min, 0.22 μm of membrane filtration degerming, the method separation that bacteriophage stoste cross according to Bacterial Plate, by from dilution
Multiple height adds in the upper strata culture medium for being mixed with 200 μ L bacterium solutions to the low direction of extension rate.Repeat the above steps 3-5 times it is repeatedly pure
Change bacteriophage, until obtaining the more uniform plaque of size, the bacteriophage as purified.And divided using double-layer agar technique measure
Potency from bacteriophage.
4th, the preservation of bacteriophage
Using liquid method of proliferating, it is 10 to be separately added into potency by 1 ratio of MOI values910 μ L of pfu/mL phagocytosis body fluid and
Bacterium number is 108Cfu/mL corresponds to culture to the 100 μ L of host strain of logarithmic phase (for bacteriophage LPSE1 in its host strain enteritis sand
It is cultivated in door Salmonella Salmonella enteritidis ATCC13076；And bacteriophage LPST10 is in its host strain mouse typhus sand
Cultivated in door Salmonella Salmonella typhimurium ATCC14028), it is inoculated in 5mL 2 × YT culture mediums, 37 DEG C are filled
Divide shaken overnight culture.Under the conditions of 4 DEG C, 8000 × g centrifugations 15min.Supernatant carefully is taken, with being dispensed after 0.22 μm of membrane filtration
To sterile vial.Two kinds of phage titers are >=10 at this time9- 80 DEG C of pfu/mL uses (containing 18% glycerine) preservation.
The morphological feature of two salmonella bacteriophages of the present invention：Two kinds of bacteriophages can be formed on solid plate
Larger bright plaque, edge clear rule.Its pnagus medius LPSE1 can be formed with 2mm plaques, and bacteriophage LPST10 exists
Diameter is in 0.5mm or so on solid plate.
The Salmonella enteritidis bacteriophage LPSE1 of purifying has been sent to China typical culture collection center preservation, classification life
Name：Salmonella enteritidis bacteriophage (Salmonella enteritidis bacteriophage) LPSE1 preserving numbers are：
CCTCC NO：M 2016472, preservation date are September in 2016 9 days, address：Wuhan, China Wuhan University.
The salmonella typhimurium bacteriophage LPST10 of purifying has been sent to China typical culture collection center preservation, classification
Name：
Salmonella typhimurium bacteriophage (Salmonella typhimurium bacteriophage) LPST10 preservations
Number is：CCTCC NO：M 2016473, preservation date are September in 2016 9 days, address：Wuhan, China Wuhan University.
Embodiment 2：
The measure of host range：
Experiment selects 8 kinds of salmonella reference cultures and 7 kinds of other kind bacterial strains to be bacteriophage LPSE1 and LPST10
Host's spectrum analysis, concrete operations are as follows：
Host's bacteria strain is cultivated respectively to logarithmic phase.Take the 100 above-mentioned bacterium solutions of μ L logarithmic phases.Treat that top-layer agar temperature is down to 46
DEG C or so when, take 3mL top-layer agars and bacterium solution mixing, pour on 15mL bottom agar culture mediums.Standing dries about 10min, treats
After the culture medium solidification of upper strata, each phagocytosis body fluid of 5 μ L is added dropwise, 5 μ L physiological saline is separately taken to do control group, observation overnight.
The results are shown in Table 1, and " ++++" " +++ " " ++ " "+" "-" represents completely limpid respectively in table；It clarifies but has dimly
Dim background；Clarifying area is substantially muddy；Multiple independent patches；Without clarification phenomenon, but may pipette tips contact position due to
There are one flecks for contact.
As a result it shows：The salmonella of a variety of different serotypes of bacteriophage LPSE1 and LPST10 cleavable, cleavable enteritis
Salmonella ATCC 13076, Bacterium enteritidis SJTUF 10978, Bacterium enteritidis SJTUF 10984, mouse typhus sramana
Salmonella ATCC 14028, salmonella typhimurium ATCC13311, moscow' paratyphi B CMCC 50094,6 kinds of cleavable
Different salmonellas, shows broad spectrum activity；There are splitting action, cleavage rate 40% to 6 plants in 15 plants of collected bacteriums.
But all salmonella bacteriophages cannot crack staphylococcus aureus, Listeria, vibrio parahaemolytious, Escherichia coli, tool
There is kind of a specificity among genus.
The measure of 1 bacteriophage LPSE1 and LPST10 host range of table
Embodiment 3：
The Electronic Speculum observation of bacteriophage
Bacteriophage stoste is through 50, after 000 × g low temperature ultracentrifugations, is resuspended in 1mL SM buffer solutions, it is small that ice bath stands 1-2
When.50 μ L samples (10 are taken respectively8-1010Pfu/mL) and 50uL volume fractions 2%, the phosphotungstic acid (phosphotun of pH 7.0
Gstic acid, PTA) it is added dropwise on sealed membrane.Copper mesh is beaten easily, is placed in sample drop and staticly settles 5-10min, it is extra to suck
Liquid.Copper mesh is taken out, stands 5min, it, will with tweezers when left and right will be dried to the suspension on copper mesh but not be completely dried
Copper mesh is placed in phosphotungstic acid (PTA) dyestuff and sucks surplus liquid after dyeing 3min, and naturally dry is to being completely dried, in transmission electron microscope
The lower observation morphology of phages, and measure its size with software Digital Micrograph Demo 3.9.1.
As a result as seen in figs. 5-6, bacteriophage LPSE1 heads after purified in icosahedral structure of virus, it is purified after bite
Thalline LPSE1 heads are in icosahedral structure of virus, head diameter about 70nm, bacteriophage tail containing shrinkage, the wide 16.6nm of tail, tail length
116.6nm。
Bacteriophage LPST10 heads are in icosahedral structure of virus, head diameter about 83.26nm, bacteriophage tail containing shrinkage, tail
Wide 10.9nm, tail length 144.89nm.Bacteriophage LPSE1 and LPST10 meet myovirus section (Myoviridae) feature.
Embodiment 4：
The drafting of bacteriophage growth curve
1st, the host strain bacterium solution of logarithmic growth early stage is prepared：
For bacteriophage LPSE1, host strain is Bacterium enteritidis ATCC13076 (present invention or referred to as 13076)；It is right
In bacteriophage LPST10, host strain is salmonella typhimurium ATCC14028 (present invention or referred to as 14028)；It is thin to be free of
2 × YT liquid of bacterium returns to zero for blank, measures its OD600It is worth for 0.4-0.5, calculating bacterial concentration.
2nd, above-mentioned diluted host strain bacterium solution 1mL is taken, is added in the ratio of optimal multiplicity of infection (MOI=0.01)
10810 μ L of pfu/mL bacteriophages, mixing, 37 DEG C incubate after 15min at 4 DEG C, and 7000 × g centrifugation 2min are discarded supernatant, used as possible
2 × YT of 1ml liquid washs 2 times, abandons supernatant.It is resuspended in equal volume with 2 × YT culture mediums,
3rd, take 50 μ L re-suspension liquids in through 37 DEG C preheating 50mL 2 × YT culture mediums in and abundant mixing, be immediately placed in 37 DEG C
160rpm/min shaken cultivations in shaking table, start simultaneously at timing, 0 moment and every 10min sample 50 μ L, 13,000 × g from
Heart 30sec, at once 20 μ L of Aspirate supernatant make gradient dilution in 180 μ L 2 × YT culture mediums.By aforesaid operations at interval of 10 points
Clock selects suitable dilution gradient, measures phage titer.
As a result as shown in Figure 7 and Figure 8, bacteriophage LPSE1 is about 30min in 0-30min bacteriophage incubation periods, 30~
110min is in burst times, and pyrolysis time is about 90min, burst size 94pfu/cfu.And bacteriophage LPST10 is in 0-30min
Bacteriophage incubation period is about 30min, and 30~60min is in burst times, and pyrolysis time is about 50min, burst size 101pfu/
cfu。
Embodiment 5：
Bacteriophage and bacteriophage composition split the evaluation of bacterium ability
1st, salmonella bacterium number and OD are measured600The relationship of value and measure phage titer.
For bacteriophage LPSE1, inoculation Bacterium enteritidis ATCC13076；For bacteriophage LPST10, it is inoculated with mouse typhus
Salmonella ATCC14028.Culture 8 hours or so takes 200 μ L to measure its OD to logarithmic phase600Value, and its bacterium number is measured simultaneously.
It determines in the OD600Under the conditions of value, the order of magnitude of contained bacterium number.
The mixed liquor of two kinds of salmonellas is with Bacterium enteritidis ATCC13076 (a concentration of 108) and mouse typhus cfu/mL
Salmonella ATCC14028 (a concentration of 108Cfu/mL) by volume 1:1(v:V) ratio mixing gained mixed liquor.
Measure phage titer：Suitable gradient is diluted to, potency is measured by double-layer agar technique.
2nd, bacteriophage splits the drafting of bacterium power curve
Phagocytosis body fluid LPSE1, LPST10 gradient dilution is taken to 105Pfu/mL, respectively according to MOI=10,1,0.1,0.01,
0.001 adds in the phagocytosis body fluid of 100 μ l corresponding concentrations after gradient dilution to experimental group, and add in 100 μ L logarithmic phase Salmonellas
Bacterium bacterium solution mixing, a concentration of the 10 of the salmonella bacterium solution8cfu/mL。
To 100 different μ L hosts bacterium solutions, (bacterium in host's bacterium solution is dense with 100 μ L of MOI=10 additions for bacteriophage composition
Spend is 108Cfu/mL in), the bacteriophage composition is LPSE1：LPST10=1：1(pfu/pfu)；When host's bacterium solution is
During 13076 and 14028 mixture, 13076 and 14028 ratio is 1:1(cfu/cfu).
The another blank group (Blank) that sets adds in 200 μ L 2 × YT culture mediums；
Control group (negative control)：100 μ L logarithmic phase detection of Salmonella bacterium solutions (OD600It is 0.4 or so) and 100 μ L
Culture medium；
Bacteriophage blank group (only contain phages)：Only add in phagocytosis body fluid (a concentration of 109pfu/mL)100μ
L and 100 μ L 2 × YT culture mediums.
Microplate reader is set：λ=600nm, Settle time：20ms, T=37.0 DEG C measure OD at interval of 1h600Change
Change.In continuous mode, with cyclotron oscillation device (orbital shaker TS-2) in 37 DEG C of mixing samples, control rotating speed is
160rpm。
2 bacteriophage LPSE1 of table splits bacterium ability under different MOI values
3 bacteriophage LPST10 of table splits bacterium ability under different MOI values
As a result as shown in table 2-3, in 6h, compared with the control group, bacteriophage LPSE1 and LPST10 is under different MOI values
Show good bacteriostasis.MOI values are higher, OD600Value decline is faster, is kept low, the antibacterial work shown
With more apparent.For bacteriophage LPSE1, work as MOI<When 1, the OD in 0-2 hours600Value raising, after 2 hours, OD600Value starts
Decline and be maintained at the level less than 0.1.And as MOI >=1, OD600Value was always held at reduced levels, sramana in 6 hours
Salmonella growth is constantly subjected to inhibit.
For bacteriophage LPST10, as MOI >=1, in 6 hours, experimental group Salmonella growth is constantly subjected to significantly
Inhibit.And work as MOI<When 1, after 2 hours, the salmonella quantity of experimental group starts to significantly reduce and significantly lower than control group.
4 bacteriophage of table and bacteriophage composition are to the bacteriostasis (MOI=10) of Bacterium enteritidis ATCC13076
5 bacteriophage of table and bacteriophage composition are to the bacteriostasis (MOI=10) of salmonella typhimurium ATCC14028
As a result as shown in upper table 4-5, for Bacterium enteritidis ATCC13076, as addition bacteriophage LPSE1 and LPST10
(1:1) during composition, OD600Value was 0.082 after 9 hours<0.1, less than the experimental group (OD for only adding in bacteriophage LPSE1600Value
0.308) and only to add in the experimental group (OD of bacteriophage LPST10600It is worth for 0.450), much smaller than control group OD600Value 0.622,
Therefore when simultaneously add in bacteriophage LPSE1 and bacteriophage LPST10 when 9 is small it is interior have to Bacterium enteritidis AT CC13076 it is bright
Aobvious inhibition, better than only adding in single bacteriophage LPSE1 or LPST10.
And for salmonella typhimurium ATCC14028, when add in bacteriophage composition 9 it is small when after, OD600It is worth and is
0.274, less than only adding in single bacteriophage LPSE1 (OD6000.378) or LPST10 (OD it is worth for600It is worth for experiment 0.308)
Group, also below control group.Therefore when simultaneously add in bacteriophage LPSE1 and bacteriophage LPST10 when 9 is small it is interior to mouse typhus sramana
Salmonella ATCC14028 has apparent inhibition, better than only adding in single bacteriophage LPSE1 or LPST10.
6 bacteriophage of table and bacteriophage composition are to the bacteriostasis of two kinds of salmonellas ATCC13076 and ATCC14028
(MOI=10)
As a result as shown in upper table 6, bacteriophage LPSE1 and bacteriophage the LPST10 OD in 6 hours600Value respectively reaches 0.20
With 0.29, less than control group OD600Value 0.44, therefore bacteriophage LPSE1 and bacteriophage LPST10 can be to two kinds of salmonella (intestines
Scorching salmonella ATCC13076 and salmonella typhimurium ATCC14028) there is inhibiting effect.And bacteriophage group ought be added in simultaneously
It closes object and (presses 1：1 ratio adds in Salmonella enteritidis bacteriophage LPSE1 and salmonella typhimurium LPST10), after 6 hours
OD600Be worth is 0.046<0.05, far below control group 0.44, less than only addition bacteriophage LPSE1 (OD6000.20) and phagocytosis it is worth for
Body LPST10 (OD600It is worth for experimental group 0.29).Illustrate, added and can more effectively inhibited in the form of two kinds of bacteriophage compositions
The growth (Fig. 9) of two kinds of salmonellas.
Embodiment 6：
The measure of bacteriophage pH stability
The 900 μ L of buffered peptone water (BPW) of different pH (2,3,4,5,6,7,8,9,10,11,12,13) are taken, are placed in 37
DEG C water bath with thermostatic control in preheat.100 μ L bacteriophage pure culture liquid (potency 10 are added in after equalized temperature8Pfu/mL), 37 DEG C
Thermostatic effect 2h.To the end of action time, double-layer agar technique is used to measure after sample is done appropriate dilution according to preliminary result
Phage titer.
As a result as shown in figs. 10-11, bacteriophage LPSE1 is stable at 10 in pH between 4-128Pfu/mL, in pH<When 3
Phage titer is reduced to 0, and in pH=13, phage titer 13pfu/mL, potency is substantially reduced, and pH is reduced to 0 when being 14.And
For bacteriophage LPST10 when pH is between 3-13, the titer plateaus of bacteriophage is in 108Pfu/mL, bacteriophage is in pH<3 or pH>
Potency is 0. when 13
Show that bacteriophage LPSE1 stablizes in pH in 4-12, bacteriophage LPST10 stablizes in pH between 3-13, shows
Good pH stability.
Embodiment 7：
The measure of bacteriophage thermal stability
With salmonella bacteriophage LPSE1 and LPST10 stoste, it is 10 to take 100 μ L potency7The bacteriophage of pfu/mL adds in
In 2 × YT culture mediums preheated 900 μ L；Respectively at 30-80 DEG C, thermostatic effect 30/60min, abundant mixing；According to preliminary experiment
As a result, selection appropriate dilutions multiple is down flat plate, the phage titer under Different treatments is measured.
As a result as illustrated by figs. 12-13, bacteriophage LPSE1 in 30-60 DEG C of titer plateaus in 107Pfu/mL, 70 DEG C of whens, are handled
Potency starts to be reduced to 10 in 30min5Pfu/mL, 80 DEG C of processing fall to 0 after sixty minutes.Show bacteriophage LPSE1 in 30-60
It is relatively stable in DEG C.
Bacteriophage LPST10 maintains 10 in 30-60 DEG C of phage titer7Pfu/mL, the phagocytosis after 70 DEG C of processing 30min
Body potency declines 4.06log pfu/mL, and phage titer declines 4.17log pfu/mL after handling 60min.Compared with LPSE1,
Bacteriophage LPST10 declines faster at 70 DEG C.Phage titer is reduced to 0 in 80 DEG C of processing 30min.
Show that bacteriophage LPSE1 and bacteriophage the LPST10 potency between 30-60 DEG C are stablized 107Pfu/mL, table
Reveal good thermal stability.
Embodiment 8：
Under the conditions of 28 DEG C of 4 DEG C of low temperature and room temperature, bacteriophage is added in different MOI values (MOI=1, MOI=100) in milk
Fungistatic effect experiment
Experimental group measures the fungistatic effect of milk bacteriophage at 4 DEG C and 28 DEG C respectively.First by control group and experimental group
Sample stands 15min-20min under the conditions of corresponding temperature is placed in.
Corresponding host strain Bacterium enteritidis ATCC13076 is added in for bacteriophage LPSE1；
Bacteriophage LPST10 adds in host strain salmonella typhimurium ATCC14028.
Under the conditions of 4 DEG C and 28 DEG C, then experimental group is divided into 2 groups respectively, respectively with the ratio of MOI=1, takes 107cfu/
10 μ L of mL bacterium solutions are after 1mL sterilizes in milk；And another group of ratio with MOI=100, take 10510 μ L of cfu/mL bacterium solutions are in 1mL
After sterilizing in milk；
Experimental group adds in 100 μ L phagocytosis body fluid (potency 106pfu/mL).Another setting is not added with the bodies such as phagocytosis body fluid, addition
The control group of product (100 μ L) SM buffer solutions.
Two groups are taken out after cultivating 0,1,2,4,6 hour under corresponding temperature (4 DEG C and 28 DEG C), through 11,000 × g from
The heart 10 minutes takes supernatant respectively, measures phage titer；Afterwards with vortex instrument mixing, control group and experimental group host strain bacterium are measured
Several variations.
Experiment is simultaneously using phage titer and bacterium number decrement as index.
For bacteriophage LPST10 groups,
During MOI=1：
Under the conditions of 4 DEG C, in the milk for adding in bacteriophage LPST10, after 6 hours, bacterium number drops compared with initial bacterium number
Low 0.31log cfu/mL；1.07log cfu/mL are reduced compared with the control group.
Under the conditions of 28 DEG C, in the experimental group bacterium number for adding in bacteriophage LPST10 compared with being initially added bacterium number, decline
1.09log cfu/mL；3.95log cfu/mL are reduced compared to the control group, and germicidal efficiency is up to 47.9%.
During MOI=100：
Under the conditions of 4 DEG C, experimental group bacterium number declines 0.84log cfu/mL compared with initial bacterium number；It drops compared to the control group
Low 1.22log cfu/mL.
Under the conditions of 28 DEG C, in addition bacteriophage LPST10 two hours, bacterium number growth is suppressed；And after 6 hours, bacterium
Number is decreased obviously 2.05log cfu/mL, compared with the control group, reduces 5.12log cfu/mL, germicidal efficiency is up to 80.4%.It is right
In bacteriophage LPSE1 groups,
During MOI=1：
Under the conditions of 4 DEG C, experimental group declines 0.24log cfu/ than control group after bacteriophage LPSE1 handles 6h in milk
mL.After processing 2h, 4h, 6h, germicidal efficiency is respectively up to 5.8%, 5.4%, 4.4%；
At 28 DEG C, after experimental group adds in bacteriophage LPSE1, bacterium number reduces 2.02log compared with control group bacterium number
Cfu/mL, germicidal efficiency is up to 26.37%.
During MOI=100：
Under the conditions of 4 DEG C, bacterium number maintains 3.31log cfu/mL after 6 hours, basically identical with control group.
Under the conditions of 28 DEG C, bacteriophage LPSE1 added in milk bacteriophage handle 6 hours after host strain bacterium number with compareing
Group is compared to 1.02log cfu/mL are reduced, and germicidal efficiency is up to 17.11%.
Illustrate both bacteriophages in MOI=1 and 100, can effectively inhibit Bacterium enteritidis in milk
The pollution of ATCC13076 and salmonella typhimurium ATCC14028.
Embodiment 9：
Under the conditions of 28 DEG C of 4 DEG C of low temperature and room temperature, bacteriophage is tested with the fungistatic effect that different MOI values are added on ham
Leg of getting fire is cut into a diameter of 2mm in aseptic operating platform, and thickness is 1mm sizes.It is divided into control group and experimental group two
Group.4/ware of number on tablet is placed in, needs 8 wares altogether.
Experimental group measures the fungistatic effect of ham bacteriophage LPSE1 and LPST10 at 4 DEG C and 28 DEG C respectively.
Under corresponding temperature (4 DEG C and 28 DEG C), host strain is added in by 1 and 100 ratios of MOI values respectively.
Bacteriophage LPSE1 adds in corresponding host strain Bacterium enteritidis ATCC13076；
Bacteriophage LPST10 adds in host strain salmonella typhimurium ATCC14028.
And work as using MOI values as 1 ratio, it is added dropwise 10750 μ L of cfu/mL bacterium solutions；And when MOI values are 100, it is added dropwise
10550 μ L of cfu/mL bacterium solutions are stood and air-dry 15-20min to surface of ham,；
10 are added dropwise to experimental group ham750 μ L of cfu/mL phagocytosis body fluid, and the SM buffer solutions of same volume are then added dropwise in control group.
It is cultivated 0,1,2,4,6 hour under corresponding temperature (4 DEG C and 28 DEG C) respectively, corresponding tablet is taken to get angry with aseptic nipper
Leg cleans 15 minutes in 5mL buffer solutions, with supersonic cleaning machine under conditions of power is 96W.Ham is taken out, by washing lotion whirlpool
Revolve the abundant mixing of instrument 30 seconds.Suitable dilution gradient is selected, measures phage titer and control group and experimental group bacterium number respectively
Variation.
For bacteriophage LPST10 groups,
As MOI=1：
Under the conditions of 4 DEG C, after 4h, the experimental group bacterium number for adding in bacteriophage LPST10 declines 2.09log compared to control group
Cfu/sample, germicidal efficiency is up to 29.6%.
Under the conditions of 28 DEG C, bacteriophage LPST10 after 4h and 6h, bacterium number decline 1.55log pfu/sample and
0.98log pfu/sample, germicidal efficiency is up to 16.7%.
As MOI=100：
Under the conditions of 4 DEG C, the experimental group bacterium number growth for adding in bacteriophage LPST10 is significantly suppressed, and declines 0.46log
Cfu/sample compares from bacterium number is initially added, and reduces 0.23log cfu/sample.
Under the conditions of 28 DEG C, bacteriophage LPST10 after 6 hours, declines 1.52log cfu/ compared with the control group in ham
Sample, germicidal efficiency is up to 33.6%；
For bacteriophage LPSE1 groups,
As MOI=1：
Under the conditions of 4 DEG C, the experimental group bacterium number for adding in bacteriophage LPSE1 declines 0.6log cfu/ compared to control group
Sample, germicidal efficiency is up to 10.8%.
Bacterium number reduces 1.86log cfu/sample to bacteriophage LPSE1 after 6 hours in ham under the conditions of 28 DEG C.Sterilization
Efficiency is up to 28.8%.
As MOI=100：
Under the conditions of 4 DEG C, the experimental group of bacteriophage is added in, bacterium number reduces 0.35log cfu/ compared with the control group
Sample, germicidal efficiency is up to 9.3%.
Under the conditions of 28 DEG C, bacterium number reduces bacteriophage LPSE1 compared with the control group under the conditions of 28 DEG C in ham
1.74log cfu/sample, while phage titer increases 0.79log pfu/sample.
Illustrate both bacteriophages in MOI=1 and MOI=100 can respectively for Bacterium enteritidis ATCC13076 and
Salmonella typhimurium ATCC14028 effectively inhibits the pollution of both serotype salmonellas in ham.
Embodiment 10：
At ambient temperature, bacteriophage is tested with fungistatic effect of the different MOI values on romaine lettuce
The pretreatment of romaine lettuce：It is wiped 1 time after tap water wash clean, then with alcohol swab, obtains the substantially sterile life in surface
The disk of a diameter of 1.5cm is made with sterilizing drill by dish sample on sterile chopping block for romaine lettuce, later with the culture of sterilizing
Ware installs sample, is placed in illumination 20min sterilization treatments under super-clean bench ultraviolet lamp, obtains sterile romaine lettuce sample.
Sample is placed in sterile petri dish center, is kept flat, smooth experiment cut side up, with liquid-transfering gun for bacteriophage
LP SE1 take 105Cfu/mL corresponds to 10 μ L of host strain Bacterium enteritidis ATCC13076；For bacteriophage LPST10, take
105Cfu/mL corresponds to 10 μ L of host strain salmonella typhimurium ATCC14028.It is random to be added dropwise in romaine lettuce sample surface, obtain sample
The host strain of upper artificial contamination is 104Cfu/Sample in order to which host strain is made fully to be adsorbed onto sample surfaces, sample is placed in super
60min is air-dried in net platform.Simultaneously in order to ensure relative humidity of the romaine lettuce blade in plate, we put in plate one it is sterile
The scraps of paper, then certain sterile water is added dropwise on the scraps of paper.
After artificial contamination's sample, romaine lettuce sample is handled with bacteriophage, it is 10 to take 10 μ L potency6The bacteriophage of pfu/mL
It is added dropwise in the position on sample, covering dropwise addition host strain as possible, it is 10 to finally obtain initial bacteriophage on sample5pfu/
For being not added with the control group of bacteriophage, the sterile 2 × YT culture solutions of 10 μ L are added dropwise in Sample.By the culture dish lid equipped with sample
It is good, sample is placed in insulating box and is stored, experiment is repeated 3 times, at the same set 3 it is parallel.
It is sampled respectively at 0,1,2,3,4,5h, sample is put into the centrifuge tube of the 5mL of the sterile PBS buffer equipped with 2ml
In, with vortex instrument, concussion 30s, gradient dilution simultaneously count at full speed.
As a result it shows：
For bacteriophage LPSE1,
Under the conditions of MOI=1, bacteriophage LPSE1 is to the fungistatic effect of Bacterium enteritidis ATCC13076：Bacteriophage with
2 hour bacterium amounts have almost no change before salmonella effect, and when acting on 3-5h, the decrement of ATCC13076 is respectively：0.6、
1.1、2.0log cfu/cm2。
Under the conditions of MOI=10, bacteriophage LPSE1 is to the fungistatic effect of Bacterium enteritidis ATCC13076：From MOI=
10 eradicating efficacy figure can be seen that 2 hour bacterium amounts before bacteriophage acts on salmonella and have almost no change, and act on 3-5h
When, the decrement of ATCC13076 is respectively：1.2、1.7、1.7log cfu/cm2。
Under the conditions of MOI=100, bacteriophage LPSE1 is to the fungistatic effect of Bacterium enteritidis ATCC13076：LPSE1 bites
Thalline acts on 0 hour bacterium amount with salmonella ATCC13076 and reduces 0.6log cfu/cm2。
For bacteriophage LPST10,
Under the conditions of MOI=1, bacteriophage LPST10 is to the fungistatic effect of salmonella typhimurium ATCC14028：Act on 3-
During 5h, the decrement of ATCC14028 is respectively：0.4、1.4、1.7log cfu/cm2。
Under the conditions of MOI=10, bacteriophage LPST10 is to the fungistatic effect of salmonella typhimurium ATCC14028：Effect
During 3-5h, the decrement of ATCC14028 is respectively：1.1、1.9、1.7log cfu/cm2。
Under the conditions of MOI=100, bacteriophage LPST10 is to the fungistatic effect of salmonella typhimurium ATCC14028：Phagocytosis
Body LPST10 acts on 0 hour (5min) bacterium amount with ATCC14028 salmonellas and reduces 0.3log cfu/cm2, with action time
Extension elimination factor constantly rise.
The above embodiments are only the preferred technical solution of the present invention, and are not construed as the limitation for the present invention, this Shen
Please in embodiment and embodiment in feature in the absence of conflict, mutually can arbitrarily combine.The protection model of the present invention
The technical solution that should be recorded with claim is enclosed, the equivalent replacement side of technical characteristic in the technical solution recorded including claim
Case is protection domain.Equivalent replacement i.e. within this range is improved, also within protection scope of the present invention.
Claims (4)
1. a kind of salmonella phage mixture, which includes Salmonella enteritidis bacteriophage LPSE1 and mouse typhus is husky
Door Salmonella bacteriophage LPST10；The Salmonella enteritidis bacteriophage (Salmonella enteritidis
Bacteriophage) deposit number of LPSE1 is：CCTCC NO：M 2016472；The salmonella typhimurium bacteriophage
(Salmonella typhimuriumBacteriophage) deposit number of LPST10 is：CCTCC NO：M 2016473.
2. phage mixture according to claim 1, in the mixture Salmonella enteritidis bacteriophage LPSE1 and
The number ratio of salmonella typhimurium bacteriophage LPST10 is 1:1.
3. application of the phage mixture described in claim 1 in food bacteriostatic agent is prepared.
4. application of the phage mixture described in claim 1 in salmonella inhibitor is prepared.
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